The present invention relates to novel molecules and, more particularly, to methods of treating CCR5/CCR5-ligands associated diseases, such as autoimmune inflammatory diseases.
Chemokines are small (˜8-14 kDa), structurally cytokine-like, secreted proteins that regulate cell trafficking. They are produced and secreted by a wide variety of cell types in response to early inflammatory mediators, such as IL-1β or TNF-α, and in response to bacterial or viral infection. Chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or damage. They can be released by many different cell types (e.g. macrophages) and can mediate a range of pro-inflammatory effects on leukocytes, such as triggering of chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation.
Chemokines can be subdivided into four classes, the C—C, C—X—C, C and C—X3-C chemokines, depending on the location of the first two cysteines in their protein sequence. The interaction of these soluble proteins with their specific receptors, which belong to the superfamily of seven-transmembrane domain G-protein-coupled receptors (GPCRs), mediate their biological effects resulting in, among other responses, rapid increase in intracellular calcium concentration, changes in cell shape, increased expression of cellular adhesion molecules, degranulation, and promotion of cell migration.
In the last several years, the key role of chemokines as important mediators in inflammatory and autoimmune disorders and diseases has been well established. Chemokines have been indicated as important mediators in multiple sclerosis (MS), allergic responses, asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis (RA), diabetic nephropathy, pulmonary fibrosis, transplant rejection and in cancer.
Among the most prominent chemokines mentioned in disorders and diseases are the C—C chemokines: MIP-1α (Macrophage inflammatory protein 1α, CCL3) and RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted, CCL5). MIP-1α is produced by macrophages and is an activator of human granulocytes (neutrophils, eosinophils and basophils). MIP-1α induces the synthesis and release of pro-inflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-α from fibroblasts and macrophages. RANTES is secreted by peripheral blood mononuclear cells (PBMC) and is a chemotactic factor for T cells, eosinophils and basophils. RANTES plays an active role in recruiting leukocytes into inflammatory sites and in inducing proliferation and activation of certain natural killer (NK) cells.
The C—C Chemokine Receptor 5 (CCR5) is expressed on several cell types including peripheral blood-derived dendritic cells, CD34+ hematopoietic progenitor cells and certain activated/memory Th1 lymphocytes. CCR5 has several C—C chemokine ligands which include CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-113), CCL5 (RANTES), CCL11 (eotaxin) and CCL16 [Blanpain et al., Blood. (1999) 94:1899-905; Nomiyama et al., Int Immunol (2001) (8):1021-9], most of which play a central role in pathogenesis.
Thus, recent studies have demonstrated the significance of CCR5 receptor and ligands in inflammatory diseases such as Rheumatoid Arthritis (RA) and Multiple Sclerosis (MS). In RA, the specific expression of CCR5 has been demonstrated on macrophages and on most T lymphocytes in the rheumatoid synovial fluid and membrane [Pokorny et al., Ann Rheum Dis (2005) 64:487-490; Wang and Liu, Clin Exp Immunol (2003) 132:371-8; Nissinen et al., J Rheumatol (2003) 30:1928-34; Mack et al., Arthritis Rheum (1999) 42:981-8] while elevated levels of chemokines, including MIP-1α (CCL3) and RANTES (CCL5), have been shown in RA synovial fluids [Hayashida et al., Arthritis Res (2001) 3(2):118-126; Loetscher and Moser, Arthritis Res (2002) 4(4):233-236]. MS is characterized by infiltration of inflammatory cells (predominantly CD4+Th1 cells) into the central nervous system (CNS). Inflammatory T cells have been shown to migrate towards RANTES and MIP-1α as a result of over expression of their receptors (CCR5) on these cells [Zang et al., Brain (2000) 123(9):1874-1882].
Thus, the potential therapeutic value of antagonizing the CCR5/ligand axis has been demonstrated.
Various approaches for blocking CCR5 activation have been attempted, some are summarized infra.
PCT Publication No. WO05078097 discloses a multifunctional short interfering nucleic acid (multifunctional siNA) molecule that modulates the expression of genes, such as CCR5, via RNA interference (RNAi) and could potentially be useful in the treatment of any disease or condition that responds to modulation of gene expression or activity. Although RNAi are known to be highly sequence specific, the decreasing rate of gene expression and protein disappearance depends on the type of targeted cell, the rate of cell division and the protein half-life. In addition, siRNA may not completely inhibit the expression of the protein and some molecules may still be transcribed, therefore, targeting the end product (the protein target itself) rather than the genes would be preferred.
U.S. Pat. No. 6,930,174 discloses CCR5 chemokine receptor-specific monoclonal antibodies which compete for receptor binding therefore blocking natural responses by interfering with ligand-receptor interactions. The contemplated use for these antibodies include treatment and/or prevention of inflammatory diseases, including rheumatoid arthritis, viral infections including Human Immunodeficiency Viruses 1 and 2 (HIV-1 and 2), cancer and auto-immune disorders. This invention has the disadvantages of using mAb: 1) they may induce anti idiotypic responses in the host; 2) they compete for receptor binding yet they do not neutralize the ligands which may still transmit activating signals. Extracellular fragments of the receptor are contemplated for antibody production including N-terminus and the first extracellular loop (EC1). EC2 is not mentioned.
U.S. Pat. No. 20030166870 discloses a mAb which binds specifically to CCR5 (anti-CCR5) and thus inhibits CCR5 functions including CCR5 binding activity (e.g. ligand binding, including RANTES, MIP-1.alpha. and/or MIP-1.beta), signaling activity (e.g., activation of a mammalian G protein) and/or stimulation of a cellular response (e.g., stimulation of chemotaxis). This anti-CCR5 mAb could be used for therapeutic interventions of inflammatory diseases as well as for HIV-1 and 2. This invention has the disadvantages of using mAb as described hereinabove. In addition, the inventors of this application, explicitly state that both the EC2 domain and the amino terminus domain of CCR5 are important for CCR5 ligand binding as manifested by their suggested epitopes for bi-specific antibody production. Receptor chimeras were described which comprise the second extracellular domain of CCR5 yet these chimeras were CCR5/CCR2 chimeras constructed by transferring restriction fragments flanked by the common BamHI, AflII, ClaI, EcoRI, and XbaI sites between human CCR5 and human CCR2b. However, these receptors can only be membrane bound and are not suitable as soluble therapeutic receptors.
To overcome these limitations, a soluble receptor-based approach has been suggested. Basically, a soluble receptor decoy which comprises the ligand binding domain is used to sequester all ligand-mediated receptor activation. The soluble receptor based approach has few apparent advantages over mAb based therapies, first, they do not readily induce anti idiotypic responses in the host, as antibodies do; in addition, according to their nature, they bind the biologically functional determinant of the ligand (which apparently transmits activating signaling in the natural receptor) and are therefore highly effective neutralizing compounds; finally, they present minimal safety issues.
U.S. Pat. No. 6,800,729 discloses a CCR5 variant (also referred to as HDGNR10) that can be used for treating diseases including chronic infections, leukemia and T-cell mediated auto-immune diseases. This invention describes a full size CCR5 protein of 352 amino acid residues with a high degree of homology to a human MCP-1 receptor. A soluble form of the described CCR5 is suggested to bind the physiological ligands (such as MIP-1) and thus may prevent the ligands from interacting with membrane bound CCR5. This invention teaches a full size CCR5 protein which is extremely large and is not compatible with therapy due to low bioavailability and degradation.
PCT Publication No. WO05106489 discloses human CCR5 fusion proteins, which can be used for diagnostics and therapeutics of diseases associated with C—C chemokines, including inflammatory diseases, hematological disorders and cancer. This fusion protein comprises a CCR5 protein fused to a galactosidase portion. This invention also provides methods of screening for therapeutic agents using the CCR5 polypeptide. Thus compounds which bind to CCR5 and inactivate CCR5-mediated signaling are identified as potential therapeutic agents. However, the teachings of this invention do not indicate which domain should be included in the CCR5 fusion. As mentioned, CCR5 is a large protein with a few extracellular domains. Whole soluble extracellular domain is very difficult to construct and is absolutely not compatible with therapy due to its bioavailability and degradation.
There is thus a widely recognized need and it would be highly advantageous to have therapeutic modalities which target CCR5 and its ligands and that can be used in the treatment of a myriad of inflammatory and autoimmune diseases which pathogenicity involves these proteins.